RESEARCH ARTICLE

Tanshinone IIA suppresses the progression of atherosclerosis byinhibiting the apoptosis of vascular smooth muscle cells and theproliferation and migration of macrophages induced by ox-LDLBaocai Wang*, Zhenwei Ge*, Zhaoyun Cheng‡ and Ziniu Zhao

ABSTRACTThe profound inhibitory effect of Tanshinone IIA (Tan IIA) onatherosclerosis has been demonstrated, whereas the latentmechanism is not completely cleared. This study aimed to investigatethe cellular and molecular mechanisms underlying Tan IIA protectingagainst atherosclerosis. Oil Red O staining and ELISA assay showedthat Tan IIA suppressed the progress of atherosclerosis and reducedthe levels of inflammatory cytokines in serum of apolipoprotein Edeficient (ApoE–/–) mice. Flow cytometry assay revealed that Tan IIAinhibited oxidized LDL (ox-LDL)-induced apoptosis of VSMCs. MTTand transwell assay indicated that Tan IIA suppressed the ox-LDL-stimulated proliferation and migration of RAW264.7 cells. Moreover,Tan IIA ameliorated inflammatory cytokine upregulation elicited byox-LDL inRAW264.7 cells. Additionally, Tan IIA inhibited the apoptosisofVSMCsanddecreased the levelsofMMP-2,MMP-9 inApoE–/–mice.In conclusion, our study demonstrated Tan IIA suppressed theprogression of atherosclerosis by inhibiting vascular inflammation,apoptosis of VSMCs and proliferation and migration of macrophagesinduced by ox-LDL.

KEY WORDS: Atherosclerosis, Macrophages, ox-LDL, TanshinoneIIA, Vascular smooth muscle cells

INTRODUCTIONAtherosclerosis is a well-known pathological manifestation ofcardiovascular diseases belonging to chronic inflammatory disease,characterized by the formation of atherosclerotic plaques (Wanget al., 2013). Lesions occur mainly in large and medium elastic andmuscular arteries and may result in ischemia of the heart, brain, andextremities, or stroke (Fenyo and Gafencu, 2013). Among numerousgenetic and environmental causes, the deposition of modified lowdensity lipoprotein (LDL), such as oxidized LDL (ox-LDL)(Steinberg et al., 1989), the recruitment of monocyte-derivedmacrophages at the arterial subendothelial spaces (Fantuzzi andMazzone, 2007), and accumulation of vascular smooth muscle cells(VSMCs) (Gerthoffer, 2007) are the crucial elements resulting inthe development of atherosclerotic lesion.

Ox-LDL has been reported in the development of atherosclerosis(Holvoet et al., 2004). Some biological processes contribute to theatherosclerotic plaque formation and progression, including smoothmuscle cell migration and proliferation, macrophage foam cellformation, and altered expression of cytokines and growth factors(Pirillo et al., 2013). Ox-LDL significantly promotes VSMCsmigration and proliferation in intimal area through activating theERK1/2 signaling pathway transcription factors, which is a mainfeature of atherosclerotic lesions (Cutini andMassheimer, 2010; Linet al., 2016; Yang et al., 2001). In macrophages, ox-LDL is capableof targeting several scavenger receptors and induces production ofproinflammatory cytokines such as tumor necrosis factor (TNF)-α,oxidative stress, and enhances chemotaxis (Sun et al., 2009).

In atherosclerosis, VSMCs are involved in reconstruction of thearterial wall in order to maintain blood flow in affected vessels dueto atherosclerotic alteration (Chistiakov et al., 2015). VSMCs existin the media in a quiescent state in the normal blood vessel, but theymigrate into the intima following injury, and their over proliferationcan cause the constraint of normal blood flow (Johnson et al., 2011;Murry et al., 1997). VSMCs apoptosis takes place in many arterialdiseases, such as angioplasty restenosis, aneurysm formation andatherosclerosis, and is connect to atherosclerotic plaque rupture(Clarke et al., 2006; Su et al., 2015). Macrophages from circulatingblood monocytes can be involved in inflammatory response(Murray and Wynn, 2011). Macrophages absorb lots of ox-LDLunder the intima and then become foam cells, which is one of theearly signs of atherosclerotic lesions (Fenyo and Gafencu, 2013).

Tanshinone IIA (Tan IIA), a kind of traditional Chinesemedicine, is one of the main fat-soluble ingredients of RadixSalvia miltiorrhiza (also known as Danshen), and a proverbialflavonoid that has demonstrated a valid antioxidant for protectingagainst atherosclerosis (You et al., 2012; Zheng et al., 2014).Previous reports show that Tan IIA inhibits the atherosclerotic lesionin rat and rabbit through suppressing the oxidative stress andinflammation (Fang et al., 2008; Tang et al., 2011). Chen and Xu(2014) indicate that Tan IIA inhibits atherosclerosis by regulatingthe apoptosis and expression of inflammatory factors inatherosclerosis plaques. Nevertheless, the precise cellular andmolecular mechanisms by which Tan IIA protect againstatherosclerosis remained unclear. Tan IIA is able to suppress theoxidative modification of LDL into ox-LDL both in vivo and in vitro(Niu et al., 2000; Tang et al., 2007). Thus, it is presumed that TanIIA exerts the anti-atherosclerotic effect likely through inhibiting theeffect of ox-LDL on atherosclerosis.

In this study, the role of Tan IIA was explored in atheroscleroticlesion in ApoE−/− mice. The functional effect of Tan IIA onVSMCs and RAW264.7 cells was further investigated to studythe molecular mechanisms of Tan IIA protecting againstatherosclerosis.Received 10 January 2017; Accepted 10 March 2017

Department of Cardiovascular Surgery, Henan Provincial People’s Hospital,Zhengzhou 450003, People’s Republic of China.*These authors contributed equally to this work

‡Author for correspondence (zhaoyunccn@163.com)

Z.C., 0000-0002-8088-7734

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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RESULTSTan IIA inhibited atherosclerosis in ApoE–/– miceTo test whether Tan IIA alleviated atherosclerosis, the plaque area ofaortic arches and staining area of aortic roots were estimated usingOil Red O staining. It was found that the aortic arches plaque area(%) (Fig. 1A,B) and aortic root slice staining area (Fig. 1C,D) ofApoE–/– mice fed with Tan IIA were obviously reduced incomparison with the control group.

Tan IIA decreased the levels of inflammatory cytokines inserum of ApoE–/– miceThe contents of four main inflammatory cytokines (IL-1β, IL-6,MCP-1, and TNF-α) in serum of ApoE–/– mice were detected byELISA. As shown in Fig. 2A-D, the serum levels of IL-1β, IL-6,MCP-1, and TNF-α in ApoE–/– mice treated with Tan IIA weredramatically reduced compared with the control group. Theseresults indicated that Tan IIA exerted an anti-inflammatory role inatherosclerosis.

Tan IIA attenuated ox-LDL-induced apoptosis of VSMCsThe effects of Tan IIA on ox-LDL-stimulated VSMCs responseswere investigated. The role of ox-LDL in apoptosis of VSMCs wasdetected by flow cytometry assay. When VSMCs were treated withox-LDL, an obvious increase in the rate of apoptosis was observed.However, this effect was significantly decreased by Tan IIAtreatment (Fig. 3A,B). Further apoptosis-related protein levels (Bax,Bcl-2, pro-caspase-3, and Cleaved caspase-3) were determined bywestern blot. The data showed that ox-LDL notably reduced theexpression of Bcl-2 and increased the levels of Bax and cleavedcaspase-3, whereas Tan IIA reversed the effects of ox-LDL(Fig. 3C,D). These results demonstrated that Tan IIA decreasedthe apoptosis induced by ox-LDL in VSMCs.

Tan IIA inhibited the ox-LDL-induced proliferation andmigration of RAW264.7 cellsThe effects of Tan IIA on ox-LDL-induced RAW264.7 cellsresponses were determined by MTT assay and migration assay. Theresults showed that ox-LDL evidently promoted RAW264.7 cellproliferation, whereas the stimulative effect of ox-LDL onRAW264.7 cell proliferation was reversed by Tan IIA (Fig. 4A);and then the effects of Tan IIA on ox-LDL-induced cell migration

were detected by cell migration assay. The data demonstrated thatthe migration ability of RAW264.7 cells treated with ox-LDL wasdrastically increased, whereas Tan IIA abated the positive effect ofox-LDL on RAW264.7 cell migration (Fig. 4B,C). In addition, thelevels of MMP-9 and MMP-2 in RAW264.7 cells were detected bywestern blot. The data indicated that protein levels of MMP-9 andMMP-2 were markedly up-regulated by ox-LDL, while Tan IIAabolished the positive effect of ox-LDL on regulating expression ofMMP-9 and MMP-2 (Fig. 4 D,E). These results suggested that TanIIA suppressed the proliferation and migration elicited by ox-LDLin RAW264.7 cells.

Tan IIA ameliorates inflammatory cytokine upregulation inox-LDL-stimulated RAW264.7 cellsThe concentrations of four main inflammatory cytokines (IL-1β,IL-6, MCP-1, and TNF-α) in RAW264.7 cells were detected byELISA. As shown in Fig. 5A-D, ox-LDL led to a prominentaugment in the levels of IL-1β, IL-6, MCP-1, and TNF-α inRAW264.7 cells compared with the control group, while Tan IIAtreatment inhibited this effect. These results indicated that Tan IIAattenuated an inflammatory response triggered by ox-LDL throughreducing cytokine production in RAW264.7 cells.

Tan IIA inhibited the apoptosis of VSMCs and decreased thelevels of MMP-2, MMP-9 in ApoE–/– miceTo further explore whether Tan IIA could alleviate the apoptosis ofVSMCs in ApoE–/– mice, VSMCs of aortic roots were estimatedusing TMR red. Western blot was used to detect the levels ofcleaved caspase-3, MMP-2 and MMP-9. The results showedthat Tan IIA could decrease the apoptosis of VSMCs in ApoE–/–

mice (Fig. 6A,B) and Tan IIA could also decrease the levels ofMMP-2 and MMP-9 in vivo (Fig. 6C) in comparison with thecontrol group.

DISCUSSIONIllustrating the mechanisms that cause the initiation anddevelopment of atherosclerosis is vital for confirming methods tosuppress its progression before it results in clinical consequences(Doran et al., 2008). Tan IIA significantly attenuated theatherosclerotic lesion in ApoE−/− mice and inhibited theformation of foam cells in atherosclerotic lesion without affecting

Fig. 1. Effects of Tan IIA on atherosclerosis inApoE−/− mice. ApoE−/− mice at 6 weeks were gavagedwith Tan IIA (30 mg/kg) for 20 weeks and then sacrificed.(A) Representative photographs indicating theatherosclerotic plaque in aortas from ApoE–/– mice withOil Red O. (B) The atherosclerotic lesion area in the TanIIA group and control group. (C) Representativephotomicrographs of the aortic root from ApoE−/− micestained with Oil Red O. (D) The atherosclerotic lesionwas quantified as the fraction area in Tan IIA group andcontrol group. In B and D, n=8, *P<0.05, error barsindicate that data were expressed as the mean±s.d.

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the lipid levels of serum (Tang et al., 2011). Recently a reportpresented by Warnatsch et al. indicated that IL-1α, IL-1β and IL-6were elevated in the plasma of ApoE-deficient animals after8 weeks on high fat diet (HFD) (Warnatsch et al., 2015). Inaccordance with our study, Tan IIA treatment significantly reducedthe atherosclerotic lesion area through inhibiting the expressions ofadhesion molecules and inflammatory cytokine secretion in serum,including IL-1β, IL-6 and MCP-1, as well as TNF-α.

Many researches have indicated that lots of cell types, such asendothelial cells, lymphocytes, macrophages and smooth musclecells (SMCs), are connected to the formation of atheroscleroticlesions (Lusis, 2000). Most atherosclerotic plaque caps consist ofVSMCs, by which the stability of plaque is maintained (Clarkeet al., 2006). Apoptosis of VSMCs was observed in unstableplaques (Su et al., 2015). To further confirm the protection of TanIIA on VSMCs, its effect on of VSMCs apoptosis was investigated.

Fig. 2. Tan IIA suppresses expression of inflammatory cytokines inserum of ApoE–/– mice. ApoE−/− mice at 6 weeks were gavaged withTan IIA (30 mg/kg) for 20 weeks and then sacrificed. (A-D) Theconcentrations of IL-1β, IL-6, MCP-1, and TNF-α in serum of ApoE–/–

mice treated with Tan IIA were significantly decreased by ELISA assay.n=8, *P<0.05, error bars indicate that data were expressed as themean±s.d.

Fig. 3. Tan IIA inhibits ox-LDL-induced apoptosis of VSMCs through regulating the apoptosis-related protein levels. VSMCs were treated with ox-LDL(50 μg/ml) or ox-LDL+Tan IIA (40 or 80 μM) for 24 h. (A,B) Flow cytometry showed that Tan IIA reversed ox-LDL-induced apoptosis in VSMCs. (C,D)Western blotdisplayed that Tan IIA overturned the effects of ox-LDL on the expression of Bcl-2, Bax and cleaved caspase-3. n=4, *P<0.05, error bars in B and D indicate thatdata were expressed as the mean±s.d.

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Our study showed ox-LDL significantly induced apoptosis ofVSMCs, while Tan IIA inhibited ox-LDL-induced apoptosis,suggesting that Tan IIA may suppress the progress ofatherosclerosis in a concentration-dependent manner. Results fromprevious studies found that Tan IIA decreased Bax protein level inatherosclerosis plaques and moderately increased Bcl-2 proteinlevels, and finally induced inhibition of atherogenesis (Xu et al.,2011). Moreover, our study also indicated that Tan IIA significantlysuppressed the Bax and Cleaved-caspase-3 up-regulation andreversed the Bcl-2 down-regulation in VSMCs treated with ox-LDL, which resulted in a reduced ratio of Baxl/Bcl-2, thusconcluding that Tan IIA suppressed atherogenesis throughregulating expression of apoptosis-related protein in VSMCs.It is certain that cell proliferation of vascular cells is one of the

mechanisms for atherosclerosis plaque growth (Andrés et al., 2012).Macrophages in the artery wall are the main cells in atherosclerosis,with the quantity and phenotype of these cells in plaques affectingdisease progression (Moore et al., 2013). Our study suggested thatox-LDL induced proliferation and migration of RAW264.7 cells,while Tan IIA reversed the inductive effect of ox-LDL onRAW264.7 cell proliferation and migration. MMP-2 and MMP-9are equal members of the MMP family. MMPs are expressed by andaround forming blood vessels, and degrading extracellular matrix isa significant role of MMPs. MMP-2 and MMP-9 are also involvedin local inflammatory cell infiltration in plaques and result indamage to the vessel wall and intimal defense function decline

(Chen and Xu, 2014). Our data indicated that Tan IIA significantlydecreased the levels of MMP-2 andMMP-9 in RAW264.7 cells pre-treated with ox-LDL.

MCP-1 and IL-6 are important cytokines in the physical andpathological processes, however IL-6 is a pleiotropic cytokinewhich can be both anti-inflammatory and pro-inflammatory.Moreover, pro-inflammatory cytokines TNF-α and IL-1β werealso lessened by Tan IIA in RAW264.7 cells treated with LPS in adose-dependent manner (Fan et al., 2016). In line with this, ourstudy demonstrated that Tan IIA inhibited the ox-LDL-induced-upregulation of IL-1β, IL-6, MCP-1 as well as TNF-α in RAW264.7cells. Thus, it is speculated that Tan IIA inhibited atherosclerosis byregulating macrophage expression of proinflammatory cytokinesand chemokine in macrophages.

Our research verified that Tan IIA significantly exerted aninhibitory effect on the initiation and progression of atherosclerosisin ApoE–/– mice through suppressing the expression of adhesionmolecules and inflammatory cytokines secretion in serum. Tan IIAattenuates ox-LDL-induced apoptosis of VSMCs, with expressionchanges of Bax, Bcl-2 and cleaved caspase-3. Tan IIA suppressed theox-LDL-induced proliferation and migration of RAW264.7 cells,accompanied by expression changes of MMP-2 and MMP-9.Additionally, our study showed that Tan IIA inhibitsoverexpression of TNF-a, IL-1β, IL-6, and MCP-1 in RAW264.7cells treated with ox-LDL. However, the molecular mechanisms bywhichTan IIAweakens atherosclerosis should be further investigated.

Fig. 4. Tan IIA inhibited the ox-LDL-induced proliferation and migration of RAW264.7 cells. RAW264.7 cells were treated with ox-LDL (50 μg/ml) or Tan IIA(40 or 80 μM). (A) MTT was used to measure the effect of ox-LDL and Tan IIA on cell proliferation. (B,C) Transwell chamber was performed to detect the effect ofox-LDL and Tan IIA on cell migration after treatment with ox-LDL or (ox-LDL+Tan IIA) for 24 h. (D,E) Western blots were carried out to determine the levels ofMMP-9 and MMP-2 after treatment with ox-LDL or (ox-LDL+Tan IIA) for 24 h in RAW264.7 cells. n=4, *P<0.05, error bars in A, C and E indicate that data wereexpressed as the mean±s.d.

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In conclusion, all data suggest that Tan IIA suppresses the progressionof atherosclerosis by inhibiting the apoptosis of VSMCs and theproliferation and migration of macrophages induced by ox-LDL,which provides new insight into the molecular mechanisms throughwhich Tan IIA may ameliorate atherosclerosis.

MATERIALS AND METHODSPreparation of ox-LDLNative LDL was purchased from Sigma-Aldrich (Shanghai, China). For theproduction of ox-LDL, 200 µg/ml LDL was exposed to 20 µM CuSO4 inphosphate-buffered saline (PBS; Keyi, Hangzhou, China) for oxidation for

Fig. 5. Tan IIA ameliorates inflammatory cytokine upregulation inox-LDL-stimulated RAW264.7 cells. RAW264.7 cells were treated withox-LDL (50 μg/ml) or (ox-LDL+Tan IIA) (40 or 80 μM) for 24 h. (A-D) Theconcentrations of inflammatory cytokines (IL-1β, IL-6, MCP-1, andTNF-α) in RAW264.7 cells treated with ox-LDL or (ox-LDL+ Tan IIA) weredetected by ELISA. n=4, *P<0.05, error bars indicate that data wereexpressed as the mean±s.d.

Fig. 6. Tan IIA reduced the apoptosis rate of VSMCs and decreased thelevels of MMP-2 and MMP-9 in ApoE−/− mice. (A) TUNEL assay showingdecrease in TUNEL-positive cells in ApoE−/− mice treated with Tan IIAagainst to control group. (B,C) Western blots confirming the levels ofcleaved caspase-3 or that the migration-related proteins MMP-2 and MMP-9 decreased when treated with Tan IIA in ApoE−/−mice, *P<0.05, error barsindicate that data were expressed as the mean±s.d. (n=8).

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20 h at 37°C and the oxidative reactions were terminated with 40 µMbutylhydroxytoluene in ethanol. Furthermore, ox-LDL was dialyzed againstculture medium and sterile filtered.

Generation of experimental atherosclerosis mice modelAll animal procedures were approved by the Ethics Committee of HenanProvince People’s Hospital. ApoE−/− mice with a C57BL/6J backgroundwere purchased from Beijing Biocytogen (Beijing, China). All animals werekept on a regular dark/light cycle, with unrestricted access to water andstandard chow (Specialty Feeds, Glen Forrest, WA, Australia) underpathogen-free conditions.

Sixteen male ApoE−/−mice at the age of 6 weeks were randomly dividedinto two groups of eight animals each: control group (NC) and Tan IIA(30 mg/kg) group. Mice in Tan IIA groups were gavaged with Tan IIA(30 mg/kg) suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na)daily for 20 weeks, whereas mice in control groups were gavaged with 0.5%CMC-Na. After 20 weeks, the mice were killed by an i.p. injection ofsodium pentobarbital (100 mg/kg; Euthatal, Sigma-Aldrich, Castle Hill,NSW, Australia).

Cytokine detectionThe levels of IL-1β, IL-6, MCP-1, and TNF-α in serum of ApoE–/– micewere determined by ELISA using the detection kit (eBioscience, San Diego,CA, USA) following manufacturer’s instructions. Absorbance at 450 nmwas evaluated using microplate reader. RAW264.7 macrophages weregrown in 96-well plates. After treatment with ox-LDL (50 μg/ml) and TanIIA (40 and 80 μM) for 24 h, cell-free supernatants were collected,centrifuged, and assayed for IL-1β, IL-6, MCP-1, and TNF-α by ELISA(eBioscience) according to the manufacturer’s instructions.

Tissue preparation and Oil Red O stainingAfter sacrifice, proximal aortae were rapidly removed from the mice,cleaned of pericardial fat under a dissecting microscope and fixed in 10%formalin. The aortas were kept at 80°C for subsequent analysis. The aorticsection was immersed in 60% isopropanol (Aladdin, Shanghai, China) for25 s and then stained with Oil Red O (Sigma-Aldrich) for 8 min. 60%isopropanol was used to remove the redundant dye for 10 s again. Sectionsof the aortic root were soaked in 60% isopropanol for 30 s and then stainedwith Oil Red O for 20 min. Washed sections were counterstained withMeyer’s hematoxylin solution (Wako Pure Chemical Industries, Osaka,Japan). The entire inner surface of aortic intima and sections of aortic rootswere photographed, and positive lesions were analyzed using Image Jsoftware (NIH, Bethesda, MD, USA).

Primary aortic smooth muscle cells isolation and cultureMouse primary VSMCs were isolated from normal aorta of male mice bycombined collagenase and elastase digestion method, as previouslydescribed (Sedding et al., 2005). VSMCs were cultured in Dulbecco’smodified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA)supplemented with 10% fetal bovine serum (FBS; GIBCO, Carlsbad, CA,USA), maintained in a 5% CO2 atmosphere at 37°C, and used for four toeight passages throughout this study.

Analysis of apoptosis in VSMCsApoptosis was analyzed by flow cytometry assay. The cells were harvestedand washed twice with cold phosphate-buffered saline (PBS). Then cells(1×106) from each sample were processed with the Annexin V-FITC/PIapoptosis detection kit (BD Biosciences, San Jose, CA, USA) according tosupplied protocols at indicated times. The caspase expression was assessedby flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Inaddition, TUNEL assay was used for the in situ cell death detection kit,TMR red, was used as described by the manufacturer (Roche Diagnostics).

RAW264.7cell cultureThe murine macrophage cell line RAW264.7 was obtained from CellCulture Center of Chinese Academy of Medical Sciences (Beijing, China).Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 10% HI-fetal bovine serum (FBS), 100 U/mlpenicillin, and 100 μg/ml streptomycin and maintained in a 5% CO2

atmosphere at 37°C. The cells were treated with different concentrations ofTan IIA (at 40 and 80 μM) or ox-LDL 50 μg/ml for 24, 48 or 72 h forfurther experiments.

RAW264.7 cell proliferation assayViable RAW264.7 cells were determined by MTT at the indicated times.Briefly, the cells were seeded at density of 2.0×103 cells/well into 96-wellplates (Corning Costar, Corning, NY, USA) in DMEM supplementedwith 10% FBS and cultured for 24 h. Following treatment with ox-LDL(50 μg/ml) and Tan IIA (40 and 80 μM), 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA)solution (5 mg/ml in ddH2O) were added to each well at indicated times(24, 48 and 72 h). The plates were incubated for a further 3∼4 h at 37°C.Intracellular formazan crystals were dissolved by the addition of 100 µl ofdimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) to each well.Cell proliferation was determined by measuring the absorbance at 490 nmby a spectrophotometer (Multiskan MK3; Thermo Fisher Scientific,Waltham, MA, USA).

Migration assayThe migration rate of RAW264.7 cells was determined using a Transwell®

chamber (Greiner; Monroe, NC, USA) with 8-µm pore filters. Tan IIAdissolved in DMEMmedium containing 0.1% FBSwas added in the bottomchamber. RAW264.7 cells (5×104 cells/well) suspended in 100 µl ofDMEM containing 0.1% BSAwas added to the upper chamber. After 5 h ofincubation, the lower side of the filter was washed with PBS. A cotton swabwas used to remove the remaining cells on the upper side of the membrane.Cells on both side of the membrane were fixed and stained with DiffQuickstaining kit (Baxter Healthcare Corp., CA, USA). Cells migrating into thelower chamber were counted in five randomly selected microscopic fields(magnification, 200×).

Western blotProtein samples were isolated from cultured cells using a total proteinextraction kit (Kaiji Biological, Inc., Nanjing, China). The proteinconcentration was detected by a NanoDrop 2000 spectrophotometer(Thermo Fisher Scientific, USA). 50 µg of proteins were separated in10% SDS-PAGE, and transferred onto polyvinylidene difluoridemembrane (PVDF; Millipore, Billerica, MA, USA). Followingblocking for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% BSA,the membranes were incubated overnight with specified primaryantibody at 4°C. PVDF membranes were washed in TBST andincubated with secondary antibody (1:5000; Santa-CruzBiotechnology, Inc.) labeled with HRP and detected by ECL. Theproteins were detected by ECL chemiluminescence detection system(Pierce, Rockford, IL, USA). The signal intensity was determinedby Image J software (NIH, USA). Antibodies used in this studyare Bax (1:10,000; CST Inc., Danvers, MA, USA), Bcl-2(1:5000;CST), Pro-caspase-3 (1:5000; CST), Cleaved-caspase-3 (1:5000;CST), MMP-9 (1:5000; CST), MMP-2 (1:5000; CST) and β-actin(1:2500; CST).

Statistical analysisDatawere presented as mean±s.d. at least three experiments. The differencesbetween different groups were analyzed using student’s t-test or analysis ofvariance (ANOVA). P<0.05 was considered statistically significant.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsThisworkwas conceivedanddesignedbyZ.C.andB.W.Theexperimentswere carriedby Z.G. Data were interpreted by Z.Z. The manuscript was prepared by B.W. and Z.C.

FundingThis research received no specific grant from any funding agency in the public.

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ReferencesAndres, V., Pello, O. M. and Silvestre-Roig, C. (2012). Macrophage proliferationand apoptosis in atherosclerosis. Curr. Opin. Lipidol. 23, 429-438.

Chen, Z. and Xu, H. (2014). Anti-inflammatory and immunomodulatory mechanismof tanshinone IIA for atherosclerosis. Evid. Based. Complement. Alternat. Med.2014, 267976.

Chistiakov, D. A., Orekhov, A. N. and Bobryshev, Y. V. (2015). Vascular smoothmuscle cell in atherosclerosis. Acta. Physiol. 214, 33-50.

Clarke, M. C., Figg, N., Maguire, J. J., Davenport, A. P., Goddard, M., Littlewood,T. D. and Bennett, M. R. (2006). Apoptosis of vascular smooth muscle cellsinduces features of plaque vulnerability in atherosclerosis. Nat. Med. 12,1075-1080.

Cutini, P. H. and Massheimer, V. L. (2010). Role of progesterone on the regulationof vascular muscle cells proliferation, migration and apoptosis. Steroids 75,355-361.

Doran, A. C., Meller, N. and McNamara, C. A. (2008). Role of smooth muscle cellsin the initiation and early progression of atherosclerosis. Arterioscler. Thromb.Vasc. Biol. 28, 812-819.

Fan, G., Jiang, X., Wu, X., Fordjour, P. A., Miao, L., Zhang, H., Zhu, Y. and Gao,X. (2016). Anti-inflammatory activity of tanshinone IIA in LPS-stimulatedRAW264.7 macrophages via miRNAs and TLR4-NF-kappaB pathway.Inflammation 39, 375-384.

Fang, Z. Y., Lin, R., Yuan, B. X., Yang, G.-D., Liu, Y. and Zhang, H. (2008).Tanshinone IIA downregulates the CD40 expression and decreases MMP-2activity on atherosclerosis induced by high fatty diet in rabbit. J. Ethnopharmacol.115, 217-222.

Fantuzzi, G. andMazzone, T. (2007). Adipose tissue and atherosclerosis: exploringthe connection. Arterioscler. Thromb. Vasc. Biol. 27, 996-1003.

Fenyo, I. M. and Gafencu, A. V. (2013). The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis.Immunobiology 218, 1376-1384.

Gerthoffer, W. T. (2007). Mechanisms of vascular smooth muscle cell migration.Circ. Res. 100, 607-621.

Holvoet, P., Kritchevsky, S. B., Tracy, R. P., Mertens, A., Rubin, S. M., Butler, J.,Goodpaster, B. and Harris, T. B. (2004). The metabolic syndrome, circulatingoxidized LDL, and risk of myocardial infarction in well-functioning elderly people inthe health, aging, and body composition cohort. Diabetes 53, 1068-1073.

Johnson, J. L., Dwivedi, A., Somerville, M., George, S. J. and Newby, A. C.(2011). Matrix metalloproteinase (MMP)-3 activates MMP-9 mediated vascularsmooth muscle cell migration and neointima formation in mice. Arterioscler.Thromb. Vasc. Biol. 31, e35-e44.

Lin, J., Zhou, S., Zhao, T., Ju, T. and Zhang, L. (2016). TRPM7 channel regulatesox-LDL-induced proliferation and migration of vascular smooth muscle cells viaMEK-ERK pathways. FEBS. Lett. 590, 520-532.

Lusis, A. J. (2000). Atherosclerosis. Nature 407, 233-241.Moore, K. J., Sheedy, F. J. and Fisher, E. A. (2013). Macrophages inatherosclerosis: a dynamic balance. Nat. Rev. Immunol. 13, 709-721.

Murray, P. J. and Wynn, T. A. (2011). Protective and pathogenic functions ofmacrophage subsets. Nat. Rev. Immunol. 11, 723-737.

Murry, C. E., Gipaya, C. T., Bartosek, T., Benditt, E. P. and Schwartz, S. M.(1997). Monoclonality of smooth muscle cells in human atherosclerosis.Am. J. Patholy. 151, 697-705.

Niu, X.-L., Ichimori, K., Yang, X., Hirota, Y., Hoshiai, K., Li, M. and Nakazawa, H.(2000). Tanshinone II-A inhibits low density lipoprotein oxidation in vitro. Free.Radic. Res. 33, 305-312.

Pirillo, A., Norata, G. D. and Catapano, A. L. (2013). LOX-1, OxLDL, andatherosclerosis. Mediators. Inflamm. 2013, 152786.

Sedding, D. G., Hermsen, J., Seay, U., Eickelberg, O., Kummer,W., Schwencke,C., Strasser, R. H., Tillmanns, H. and Braun-Dullaeus, R. C. (2005). Caveolin-1facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo.Circ. Res. 96, 635-642.

Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. and Witztum, J. L.(1989). Beyond cholesterol. Modifications of low-density lipoprotein that increaseits atherogenicity. N. Engl. J. Med. 320, 915-924.

Su, G., Sun, G., Liu, H., Shu, L., Zhang, J., Guo, L., Huang, C. and Xu, J. (2015).Niacin suppresses progression of atherosclerosis by inhibiting vascularinflammation and apoptosis of vascular smooth muscle cells. Med. Sci. Monit.21, 4081-4089.

Sun, L., Ishida, T., Yasuda, T., Kojima, Y., Honjo, T., Yamamoto, Y., Yamamoto,H., Ishibashi, S., Hirata, K. and Hayashi, Y. (2009). RAGE mediates oxidizedLDL-induced pro-inflammatory effects and atherosclerosis in non-diabetic LDLreceptor-deficient mice. Cardiovasc. Res. 82, 371-381.

Tang, F., Wu, X., Wang, T., Wang, P., Li, R., Zhang, H., Gao, J., Chen, S., Bao, L.,Huang, H. et al. (2007). Tanshinone II A attenuates atherosclerotic calcification inrat model by inhibition of oxidative stress. Vascul. Pharmacol. 46, 427-438.

Tang, F.-T., Cao, Y., Wang, T.-Q., Wang, L.-J., Guo, J., Zhou, X.-S., Xu, S. W., Liu,W.-H., Liu, P.-Q. and Huang, H.-Q. (2011). Tanshinone IIA attenuatesatherosclerosis in ApoE(-/-) mice through down-regulation of scavengerreceptor expression. Eur. J. Pharmacol. 650, 275-284.

Wang, X.-H., Wang, F., You, S.-J., Cao, Y.-J., Cao, L.-D., Han, Q., Liu, C.-F. andHu, L.-F. (2013). Dysregulation of cystathionine gamma-lyase (CSE)/hydrogensulfide pathway contributes to ox-LDL-induced inflammation in macrophage. Cell.Signal. 25, 2255-2262.

Warnatsch, A., Ioannou, M., Wang, Q. and Papayannopoulos, V. (2015).Inflammation. Neutrophil extracellular traps license macrophages for cytokineproduction in atherosclerosis. Science 349, 316-320.

Xu, S., Little, P. J., Lan, T., Huang, Y., Le, K., Wu, X., Shen, X., Huang, H., Cai, Y.,Tang, F. et al. (2011). Tanshinone II-A attenuates and stabilizes atheroscleroticplaques in apolipoprotein-E knockout mice fed a high cholesterol diet. Arch.Biochem. Biophys. 515, 72-79.

Yang, C.-M., Chien, C.-S., Hsiao, L.-D., Pan, S.-L., Wang, C.-C., Chiu, C.-T. andLin, C.-C. (2001). Mitogenic effect of oxidized low-density lipoprotein on vascularsmooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway.Br. J. Pharmacol. 132, 1531-1541.

You, Z., Xin, Y., Liu, Y., Han, B., Zhang, L., Chen, Y., Chen, Y., Gu, L., Gao, H. andXuan, Y. (2012). Protective effect of Salvia miltiorrhizae injection on N(G)-nitro-D-arginine induced nitric oxide deficient and oxidative damage in rat kidney. Exp.Toxicol. Pathol. 64, 453-458.

Zheng, S., Ren, Z., Zhang, Y. and Qiao, Y. (2014). Anti-inflammatory mechanismresearch of tanshinone II A by module-based network analysis. Biomed. Mater.Eng. 24, 3815-3824.

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RESEARCH ARTICLE Biology Open (2017) 6, 489-495 doi:10.1242/bio.024133

BiologyOpen

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